Both the detection and use of electricity/electric fields in medicine and biology are widespread and well accepted. Electrocardiography (EKG) and electroencephalography (EEG) are used to detect electrical activity in the heart and brain, respectively. Cardioversion, the application of a pulsed electric field to heart muscle, is routinely used to stop, modify, or re-start the heart's beating. Low power electric fields can be applied to bone fractures to stimulate healing. Electromyography, the application of measured electrical pulses to muscles or their associated nerves, can be used to measure muscle function and/or judge the degrees of muscle damage. In biology, electric fields have various applications and can be used, for example, to separate molecules of different sizes (electrophoresis) or different charges (isoelectric focusing), and to separate cells with different characteristics (cell sorting during flow cytometry). Electric fields can also be used to facilitate entry of new proteins or genes into living cells via a process called electroporation.
In electroporation, application of brief (on the scale of thousandths-to-millionths of a second), moderate power (kilovolt/meter) electric fields causes permeabilization (leakiness) of the cell's surface membrane which then allows entry of materials/molecules into the cell that would otherwise never gain access to the cell's interior. After the initial permeabilization of the cell membrane, the cell eventually returns to its normal “non-leaky” condition. Now, however, the cell will carry and/or utilize the materials that have been introduced into it by the electroporation. This process can be used for the introduction of genes or drugs into a cell, for example, for transdermal drug delivery (Neumann, E, Kakorin, S., and Toensing, K. (1999), Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem. Bioenerg. 48, 3-16.1999; Weaver, J. C., Vaughan, T. E., and Chizmadzhev, Y. (1999), Theory of electrical creation of aqueous pathways across skin transport barriers. Adv. Drug Deliv. Rev. 35, 21-39), and as a therapeutic tool for the treatment of cancer using electrochemotherepy (Belehradek, M., Domenge, C., Luboinski, B., Orlowski, S., Belehradek, J. Jr., and Mir, L. M. (1993), Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer 72, 3694-700.1993; Heller, R., Jaroszeski, M. J., Glass, L. F., Messina, J. L., Rapaport, D. P., DeConti, R. C., Fenske, N. A., Gilbert, R. A., Mir, L. M., Reintgen, D. S. (1996), Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer 77, 964-71.1996; Hofmann, F, Ohnimus, H., Scheller, C., Strupp, W., Zimmermann, U., and Jassoy, C. (1999), Electric field pulses can induce apoptosis. J. Membr. Biol. 169, 103-109). Electrochemotherapy or electroporation therapy (EPT) is a method for the in vivo delivery of poorly permeable chemotherapeutic agents, such as bleomycin, to tumor cells that can be appropriately oriented between two electrodes (Dev S. B., Hofmann, G. A., Electrochemotherapy—a novel method of cancer treatment. Cancer Treat Rev 20:105-15, 1994; Hofmann et al., Electroporation therapy: a new approach for the treatment of head and neck cancer. IEE Trans Biomed Eng 46:752-9, 1999; Mir, L. M., Orlowski, S. Mechanisms of electrochemotherapy. Adv Drug Deliv Rev., 35:107-118, 1999). Both electroporation and EPT are dependent on electric effects on the plasma membrane of the cells or tissues.
Electroporation occurs with pulse durations on the order of 0.1 to 20 milliseconds (“ms”) (Dev, S. B., Rabussay, D. A., Widera, G., and Hofmann, G A (2000) IEEE Trans. Plasma Sci. 28, 206-223) with electric fields on the order of volts to low kilovolts/centimeter; however, specific conditions depend on the particular cell type and the cell suspension media. These millisecond pulses promote transient membrane poration and cell survival. Alternatively, using different electrical or cellular conditions, electroporation can cause rupture/death of cells. Although the physical nature of the pores is not well characterized, the experimental conditions that allow intracellular delivery of membrane impermeable molecules with good cell survivability are well known. Conditions for optimal electroporation depend on the waveform, the constituents of the media in which the cell is suspended, and the cell type (Weaver, J. C., Electroporation of cells and tissues, in: J. D. Bronzino (Ed.), The Biomedical Engineering Handbook, CRC and IEEE press, Boca Raton, Fla., 1995, pp. 1431-1440; Djuzenova et al., Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells. Biochim Biophys Acta, 1284:143-52, 1996). In any case, the electroporation effects of these millisecond low power applied electric fields occur only at the cell's surface membrane.
As mentioned above, electric fields and the process of electroporation have also been used for the introduction of genes into cells. The transfection of living cells with DNA is a common molecular technique used to express exogenous genes in cells for transcription studies or for therapeutic purposes in the treatment of some diseases. Known transfection methods include the incorporation of DNA into lipid vesicles for fusion with the plasma membrane, the endocytosis of DNA precipitated with calcium phosphate or dextran, the use of viral vectors that infect the cell with the gene of interest, and electropermeabilization or electroporation using pulsed electric fields that form “pores” in the plasma membrane. Some cell types, especially those that grow in suspension, can only be effectively transfected by electropermeabilization. Enhanced or optimized gene expression has been previously accomplished using classical electroporation pulses by changing the pulse duration of a long pulsed electric field (for example, within the range of 1 microsecond-20 milliseconds), changing the electric field intensity within classical electroporation range (0.1-5 kV/cm), and/or by modifying the conductivity of the buffer or media. In other transfection procedures enhanced gene expression has been accomplished by changing the concentration of DNA used in the transfection procedure, changing the physical/chemical properties during transfection (pH, ionic strength, etc), using various lipid combinations with different properties, or adding other constituents to the cell culture media or buffers to aid transfection efficiency.
Even with these known techniques, more efficient methods of introducing an agent into a cell and new methods of enhancing gene expression are still needed. These and various other needs are addressed, at least in part, by one or more embodiments of the present invention.